MALDI ImagingMass Spectrometry

Nan KleinholzMass Spectrometry and Proteomics Facility

The Ohio State UniversityProteomics Summer Workshop 2015

What is MALDI Imaging?

MALDI:Matrix Assisted Laser Desorption Ionization

Imaging:Imaging is a technique in which a sample, often a thin

tissue section, is moved in two dimensions while the mass spectrum is recorded.

Things to think about

Goals• Small molecule/lipids• Peptide/protein• Others…

Sample type• Heart, lung, liver, etc.• Biofilm• Others…

Method optimization!• Appropriate matrix• Standards

IMS Workflow

Sample Preparation

Matrix Application Analysis Processing

MALDI

Sample Preparation

Fresh Frozen (FF) is best for all IMS analysis.

BUT:Hundreds and thousands of tissue samples have been “banked” over the years as Formalin Fixed Paraffin Embedded (FFPE).

Lipids and small molecules: FF only.

Intact proteins: FF or FFPE

FFPE samples:• Tissue sectioning• Deparaffinization and Rehydration• Heat induced antigen retrieval• Histology staining on serial section (i.e. Hematoxylin and eosin)• Matrix application

FF samples:• Tissue sectioning• Washing• Histology staining on serial section• Matrix application

Cryosectioning-FF

Microtome-FFPE

Matrix Application

Wet, but not too wet.• Extraction• Delocalization

Sufficient matrix application.• Some• But not too much

Matrix choice• Works well• Doesn’t work…

And the list goes on…

Automated Matrix Application Methods

Spotted Arrays• Labcyte Portrait—uses an acoustic pico drop

method

Bruker ImagePrep

HTX Technologies SprayerTM-Sprayer

TLC sprayer Sublimation

Manual Matrix Application Methods

Typical Workflow for peptide and protein imaging

Tissue slice on slide

Wash tissue by pipette or bath

Common solvents:• Ethanol• Acetonitrile

Allow tissue to dry

Apply matrix

MALDI IMS

SA, DHA: proteinsHCCA, DHB: peptides

Apply trypsin for digestionIncubate at 37°C for 2-18 hours

Top-Down or Bottom-Up Analysis?

Top-Down: extract and ID intact proteins in images• Faster sample prep for imaging• Fewer steps means less chance for delocalization or other

error• Increase confidence in ID

Bottom-Up: sequence tryptic fragments of larger proteins• Easier to analyze larger or hydrophobic proteins• Potential for MALDI MS/MS

Tools for Protein ID post IMS Top Down Analysis for intact proteins

LC-MS of proteins

ETD MS/MS fragmentation

De-novo sequencing

Bottom-Up Analysis for tryptic peptides

Tissue microextraction

CID MS/MS fragmentation

Database searchingMatch masses of protein ID’s to IMS data

Tissue microextraction

Matrix removal

LC-MS of peptides

Matrix removal

Typical workflow for lipids and small molecule imaging

Fresh Frozen tissue samples

Wash with 50mM Ammonium formate to reduce salts

Apply appropriate matrix

Lipids: DHB, DHA, DAN, 9-AA

Small molecules: DHB, SA, HCCA, THAP

MALDI IMS

AnalysisInstrumentation in our facility with imaging capability

Bruker ultrafleXtreme MALDI-TOF-TOF Bruker solariX FTMS – MALDI and ESI

Nature Reviews Cancer 10, 639-646 (September 2010) | doi:10.1038/nrc2917

Processing

• A challenge in imaging experiments is the huge amount of generated data. A typical 2D MALDI-TOF imaging dataset contains thousands of spectra.

• Software programs allow statistical analysis and overlaying of the histology staining. (SCiLS—Bruker supported.)

H and E staining with tumor indicated

Dataset provided by SCiLS

Total mass spectrum for entire imaging run

Three masses selected as having statistical differences

For further information, please come see me in BRT 250.

Not actually me…